Cancer includes a broad range of diseases, affecting approximately one in four individuals worldwide. The severity of the adverse impact of cancer is profound, influencing medical policy and procedure as well as society generally. Because a hallmark of many types of cancer is rapid and unregulated proliferation of malignant cells, an overarching problem in improving approaches to cancer is the need for early detection and diagnosis. Numerous attempts have been made to develop accurate and reliable criteria for diagnosing the presence of a malignant condition. In particular, efforts have been directed to the use of serologically defined antigenic markers known as tumor associated antigens, which are either uniquely expressed by cancer cells or are present at markedly higher levels in subjects having a malignant condition.
However, due to the high heterogeneity of tumor associated antigen expression, for example the extreme diversity of carcinoma antigens, there is a need for additional tumor markers that are useful in cancer diagnosis. Many monoclonal antibodies reactive with carcinoma associated antigens are known (see, e.g., Papsidero, 1985 Semin. Surg. Oncol. 1:171, Allum et al., 1986 Surg. Ann. 18:41). These and other described monoclonal antibodies bind to a variety of different carcinoma associated antigens including glycoproteins, glycolipids and mucins (see, e.g., Fink et al., 1984 Prog. Clin. Pathol. 9:121; U.S. Pat. No. 4,737,579; U.S. Pat. No. 4,753,894; U.S. Pat. No. 4,579,827; U.S. Pat. No. 4,713,352). Many such monoclonal antibodies recognize tumor associated antigens that exhibit restricted expression on some but not other tumors originating in a given cell lineage or tissue type.
There are only relatively few examples of tumor associated antigens that appear to be useful for identifying a particular type of malignancy. Monoclonal antibody B72.3, for example, specifically binds to a high molecular mass (>106 Da) tumor-associated mucin antigen that is selectively expressed on a number of different carcinomas, including most if not all ovarian carcinomas and an overwhelming majority of non-small cell lung carcinomas, colon carcinomas and breast carcinomas (see, e.g., Johnston, 1987 Acta Cytol. 1:537; U.S. Pat. No. 4,612,282). Nevertheless, detection of cell-associated tumor markers such as the mucin antigen recognized by B72.3 following surgical resection of a tumor may be of limited usefulness for diagnostic screening, in which early detection of a malignant condition prior to accumulation of substantial tumor mass is preferred.
An alternative to the diagnosis of a particular type of cancer by screening surgically resected specimens for tumor associated antigens, where invasive surgery is usually indicated only after detection of an accumulated tumor mass, would be to provide compositions and methods for detecting such antigens in samples obtained from subjects by non-invasive or minimally invasive procedures. In ovarian and other carcinomas, for example, there are currently a number of soluble tumor associated antigens that are detectable in samples of readily obtained biological fluids such as serum or mucosal secretions. One such marker is CA125, a carcinoma associated antigen that is also shed into the bloodstream, where it is detectable in serum (e.g., Bast et al., 1983 N. Eng. J. Med. 309:883; Lloyd et al., 1997 Int. J. Canc. 71:842). CA125 levels in serum and other biological fluids have been measured along with levels of other markers, for example, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN mucin (STN) and placental alkaline phosphatase (PLAP), in efforts to provide diagnostic and/or prognostic profiles of ovarian and other carcinomas (e.g., Sarandakou et al., 1997 Acta Oncol. 36:755; Sarandakou et al., 1998 Eur. J. Gynaecol. Oncol. 19:73; Meier et al., 1997 Anticanc. Res. 17(4B):2945; Kudoh et al., 1999 Gynecol. Obstet. Invest. 47:52; Ind et al., 1997 Br. J. Obstet. Gynaecol. 104:1024; Bell et al. 1998 Br. J. Obstet. Gynaecol. 105:1136; Cioffi et al., 1997 Tumori 83:594; Meier et al. 1997 Anticanc. Res. 17(4B):2949; Meier et al., 1997 Anticanc. Res. 17(4B):3019).
Elevated levels of serum CA125 alone or in combination with other known indicators, however, do not provide a definitive diagnosis of malignancy, or of a particular malignancy such as ovarian carcinoma. For example, elevated CA125, CEA and SCC in vaginal fluid and serum correlate most strongly with inflammation in benign gynecological diseases, relative to cervical cancer and genital tract cancers (e.g., Moore et al., 1998 Infect. Dis. Obstet. Gynecol. 6:182; Sarandakou et al., 1997 Acta Oncol. 36:755). As another example, elevated serum CA125 may also accompany neuroblastoma (e.g., Hirokawa et al., 1998 Surg. Today 28:349), while elevated CEA and SCC, among others, may accompany colorectal cancer (Gebauer et al., 1997 Anticanc. Res. 17(4B):2939). Another marker, the differentiation antigen mesothelin, is expressed on the surfaces of normal mesothelial cells and also on certain cancer cells, including epithelial ovarian tumors and mesotheliomas. Compositions and methods pertaining to mesothelin (Chang et al., 1992 Canc. Res. 52:181; Chang et al., 1992 Int. J. Canc. 50:373; Chang et al., 1992 Int. J. Canc. 51:548; Chang et al., 1996 Proc. Nat. Acad. Sci. USA 93:136; Chowdhury et al., 1998 Proc. Nat. Acad. Sci. USA 95:669; Yamaguchi et al., 1994 J. Biol. Chem. 269:805; Kojima et al., 1995 J. Biol. Chem. 270:21984) and structurally related mesothelin related antigen (MRA; see, e.g., Scholler et al., 1999 Proc. Nat. Acad. Sci. USA 96:11531) are known in the art, including uses in cancer detection and therapies as described in WO 00/50900 and in U.S. application Ser. No. 09/513,597. Thus the compelling need for additional markers to be used, including markers useful in multi-factor diagnostic screening, is apparent. (See, e.g., Sarandakou et al., 1998; Kudoh et al., 1999; Ind et al., 1997.)
As described in greater detail below, such additional markers might be usefully provided from within the “four-disulfide core” family of proteins, which comprises a heterogeneous group of small acid- and heat-stable molecules of divergent function and which includes human epididymal four-disulfide core protein, or “HE4” (Kirchhoff et al., 1991 Biol. Reprod. 45:350-357; Wang et al., 1999 Gene 229:101; Schummer et al., 1999 Gene 238:375). The conserved spacing of eight core cysteine residues in the amino acid sequences of four-disulfide core family member polypeptides is thought to direct the folding of these molecules into a compact and stable structure. Many of the members of the four-disulfide core family are protease inhibitors; however, for some family members, including HE4, no function has yet been definitively identified. Other members of the four-disulfide core family of proteins include Wp-protein, SLP-1, and ps20, and additional members of the four-disulfide core family of proteins have been isolated from several species. These proteins share several properties, including their small size and their heat- and acid-stable structure, which is stabilized by the four-disulfide core. These proteins are made by secretory cells, and are found in mucous secretions such as seminal plasma, milk, parotid, and cervical secretions.
The prototype molecule of the four-disulfide core family, Wp-protein, is also known as the whey phosphoprotein, and has been cloned (Dandekar et al., 1982 Proc. Natl. Acad. Sci. USA 79: 3987-3991). Whey phosphoprotein is expressed in milk at approximately 1-2 mg/ml, but is not expressed by breast carcinomas, where the gene is hypermethylated. No inhibitory activity towards proteases has been found for whey phosphoprotein. However, overexpression of the gene in transgenic animals impairs development of mammary alveolar cells (Burdon et al, 1991 Mechanisms Dev. 36: 67-74), suggesting an important role for this protein during lactation. The secretory leukocyte protease inhibitor (SLP-1), another four-disulfide core family protein, was cloned from human cervix uteri, but is also present in other mucus secretions including seminal plasma and parotid secretions (Heinzel et al, 1986 Eur. J. Biochem. 160: 61-67). SLP-1 is a two domain protein of 12 kDa that is a potent inhibitor of trypsin, chymotrypsin, elastase, and cathepsin G. The crystal structure of SLP-1 complexed with chymotrypsin has been published (Grutter et al, 1988 EMBO J. 7: 345-351). These data showed that SLP-1 domains can work independently and simultaneously to inhibit different proteases, and identified a small (8 amino acids) active site in domain two that binds to chymotrypsin.
Elafin is a single domain protein member of the four-disulfide core family that was isolated from human psoriatic skin to determine the amino acid sequence of this polypeptide (Wiedow et al, 1990 J. Biol. Chem. 265: 14791-14795). Elafin is a potent inhibitor of elastase, but does not exhibit apparent inhibitory activity toward other proteases such as trypsin, chymotrypsin, cathepsin G or plasmin. The amino acid sequence of elafin shows 38% homology with the C-terminal region (domain 1) of SLP-1. The gene encoding the ps20 protein was recently isolated from smooth muscle, and the ps20 protein was expressed by transfection of the gene into mammalian cells (Larsen et al, 1998 J. Biol. Chem. 273: 4574-4584). ps20 was found to inhibit growth of carcinoma cells, and ps20 has been referred to as a growth inhibitor; however, no direct functional activity such as inhibition of proteases has been described so far for this protein.
As noted above, no protease inhibitory activity has been identified for HE4, which was initially identified in epididymal eptithelial cells, although other small acid and heat stable proteinase inhibitors have been characterized from seminal plasma, and are thought to play a role in fertility by binding to spermatozoa and regulating the interaction of spermatozoa with the extracellular matrices of the egg (Fitz et al., in Proteases and Biological Controls, Reich, E., Rifkin, D., Shaw, E. (eds.), 1975 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 737-766; Saling, 1989 Oxf. Rev. Reprod. Biol. 11: 339-388). HE4 cDNA was first isolated from human epididymis (Kirchhoff et al., 1991 Biol. Reprod. 45:350-357), and HE4 cDNA was later detected with high frequency in cDNA libraries constructed from ovarian carcinomas (Wang et al., 1999 Gene 229:101; Schummer et al., 1999 Gene 238:375).
For reasons given above, clearly there is a need for improved diagnostic markers and therapeutic targets for the detection and treatment of malignant conditions, such as carcinomas. The compositions and methods of the present invention overcome these limitations of the prior art by providing a method of screening for the presence of a malignant condition using antibodies specific for HE4 and/or HE4-related antigens to detect polypeptides that naturally occur in soluble form and/or on cell surfaces, and offer other related advantages.